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1.
BMC Med Genet ; 21(1): 9, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31914974

RESUMO

BACKGROUND: Coagulation factor XIII (FXIII) plays an essential role in maintaining hemostasis by crosslinking fibrin. Deficiency in FXIII affects clot stability and increases the risk of severe bleeding. Congenital FXIII deficiency is a rare disease. Recently, we identified a Chinese family with FXIII deficiency and investigated the pathogenesis of congenital FXIII deficiency, contributing non-coding pathogenic variants. METHODS: We performed common tests, coding sequencing by targeted next-generation sequencing (NGS), whole-genome sequencing and splice-sites prediction algorithms. The pathogenesis was investigated via minigene and nonsense-mediated mRNA decay (NMD) by experiments in vitro. RESULTS: The proband is homozygote for a novel deep intronic c.799-12G > A mutation in the F13A1 gene. Through direct sequencing of the minigenes mRNA, we found 10 bases of intron 6 insert in the mRNA of mutant minigenes mRNA. The relative expression of EGFP-F13A1 was higher by suppression of NMD in vitro. Furthermore, we found the proband with enhanced thrombin generation (TG). CONCLUSION: We reported a novel deep intronic c.799-12G > A mutation of F13A1 which produced a new acceptor site and frame shifting during translation introducing a premature termination codon. Our results support the premature termination codon triggered NMD. We need to pay attention to the position of potential alterable splicing sites while counselling and genetic test. The finding of enhanced TG indicated that we should be aware of the risk of thrombosis in patients with FXIII deficiency during replacement therapy.


Assuntos
Transtornos da Coagulação Sanguínea/genética , Deficiência do Fator XIII/genética , Fator XIII/genética , Adolescente , Adulto , Transtornos da Coagulação Sanguínea/sangue , Transtornos da Coagulação Sanguínea/patologia , Pré-Escolar , Deficiência do Fator XIII/sangue , Deficiência do Fator XIII/patologia , Feminino , Humanos , Íntrons/genética , Masculino , Mutação , Degradação do RNAm Mediada por Códon sem Sentido/genética , Linhagem , Processamento de RNA , RNA Mensageiro/genética
2.
Nucleic Acids Res ; 48(3): 1327-1340, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31879760

RESUMO

Intron retention (IR) has been proposed to modulate the delay between transcription and translation. Here, we provide an exhaustive characterization of IR in differentiated white blood cells from both the myeloid and lymphoid lineage where we observed highest levels of IR in monocytes and B-cells, in addition to previously reported granulocytes. During B-cell differentiation, we found an increase in IR from the bone marrow precursors to cells residing in secondary lymphoid organs. B-cells that undergo affinity maturation to become antibody producing plasma cells steadily decrease retention. In general, we found an inverse relationship between global IR levels and both the proliferative state of cells, and the global levels of expression of splicing factors. IR dynamics during B-cell differentiation appear to be conserved between human and mouse, suggesting that IR plays an important biological role, evolutionary conserved, during blood cell differentiation. By correlating the expression of non-core splicing factors with global IR levels, and analyzing RNA binding protein knockdown and eCLIP data, we identify a few splicing factors likely playing an evolutionary conserved role in IR regulation. Our work provides new insights into the role of IR during hematopoiesis, and on the main factors involved in regulating IR.


Assuntos
Linfócitos B/metabolismo , Hematopoese/genética , Íntrons/genética , Fatores de Processamento de RNA/genética , Processamento Alternativo/genética , Animais , Linfócitos B/citologia , Diferenciação Celular/genética , Proliferação de Células/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Silenciamento de Genes , Humanos , Ativação Linfocitária/genética , Camundongos , Degradação do RNAm Mediada por Códon sem Sentido/genética , Proteínas de Ligação a RNA/genética
3.
PLoS Comput Biol ; 15(10): e1007467, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658270

RESUMO

The somatic co-evolution of tumors and the cellular immune responses that combat them drives the diversity of immune-tumor interactions. This includes tumor mutations that generate neo-antigenic epitopes that elicit cytotoxic T-cell activity and subsequent pressure to select for genetic loss of antigen presentation. Most studies have focused on how tumor missense mutations can drive tumor immunity, but frameshift mutations have the potential to create far greater antigenic diversity. However, expression of this antigenic diversity is potentially regulated by Nonsense Mediated Decay (NMD) and NMD has been shown to be of variable efficiency in cancers. Here we studied how mutational changes influence global NMD and cytolytic immune responses. Using TCGA datasets, we derived novel patient-level metrics of 'NMD burden' and interrogated how different mutation and most importantly NMD burdens influence cytolytic activity using machine learning models and survival outcomes. We find that NMD is a significant and independent predictor of immune cytolytic activity. Different indications exhibited varying dependence on NMD and mutation burden features. We also observed significant co-alteration of genes in the NMD pathway, with a global increase in NMD efficiency in patients with NMD co-alterations. Finally, NMD burden also stratified patient survival in multivariate regression models in subset of cancer types. Our work suggests that beyond selecting for mutations that elicit NMD in tumor suppressors, tumor evolution may react to the selective pressure generated by inflammation to globally enhance NMD through coordinated amplification and/or mutation.


Assuntos
Citotoxicidade Imunológica/genética , Neoplasias/genética , Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , Evolução Biológica , Simulação por Computador , Citosol/metabolismo , Bases de Dados Genéticas , Evolução Molecular , Mutação da Fase de Leitura/genética , Humanos , Aprendizado de Máquina , Mutação/genética , Mutação de Sentido Incorreto/genética
4.
Nat Genet ; 51(11): 1645-1651, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31659324

RESUMO

Premature termination codons (PTCs) can result in the production of truncated proteins or the degradation of messenger RNAs by nonsense-mediated mRNA decay (NMD). Which of these outcomes occurs can alter the effect of a mutation, with the engagement of NMD being dependent on a series of rules. Here, by applying these rules genome-wide to obtain a resource called NMDetective, we explore the impact of NMD on genetic disease and approaches to therapy. First, human genetic diseases differ in whether NMD typically aggravates or alleviates the effects of PTCs. Second, failure to trigger NMD is a cause of ineffective gene inactivation by CRISPR-Cas9 gene editing. Finally, NMD is a determinant of the efficacy of cancer immunotherapy, with only frameshifted transcripts that escape NMD predicting a response. These results demonstrate the importance of incorporating the rules of NMD into clinical decision-making. Moreover, they suggest that inhibiting NMD may be effective in enhancing cancer immunotherapy.


Assuntos
Edição de Genes , Doenças Genéticas Inatas/genética , Imunoterapia , Neoplasias/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Mensageiro/genética , Humanos , Modelos Genéticos , Neoplasias/imunologia , Neoplasias/terapia
5.
PLoS Genet ; 15(9): e1008386, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31527909

RESUMO

Errors throughout gene expression are likely deleterious, hence genomes are under selection to ameliorate their consequences. Additional stop codons (ASCs) are in-frame nonsense 'codons' downstream of the primary stop which may be read by translational machinery should the primary stop have been accidentally read through. Prior evidence in several eukaryotes suggests that ASCs are selected to prevent potentially-deleterious consequences of read-through. We extend this evidence showing that enrichment of ASCs is common but not universal for single cell eukaryotes. By contrast, there is limited evidence as to whether the same is true in other taxa. Here, we provide the first systematic test of the hypothesis that ASCs act as a fail-safe mechanism in eubacteria, a group with high read-through rates. Contra to the predictions of the hypothesis we find: there is paucity, not enrichment, of ASCs downstream; substitutions that degrade stops are more frequent in-frame than out-of-frame in 3' sequence; highly expressed genes are no more likely to have ASCs than lowly expressed genes; usage of the leakiest primary stop (TGA) in highly expressed genes does not predict ASC enrichment even compared to usage of non-leaky stops (TAA) in lowly expressed genes, beyond downstream codon +1. Any effect at the codon immediately proximal to the primary stop can be accounted for by a preference for a T/U residue immediately following the stop, although if anything, TT- and TC- starting codons are preferred. We conclude that there is no compelling evidence for ASC selection in eubacteria. This presents an unusual case in which the same error could be solved by the same mechanism in eukaryotes and prokaryotes but is not. We discuss two possible explanations: that, owing to the absence of nonsense mediated decay, bacteria may solve read-through via gene truncation and in eukaryotes certain prion states cause raised read-through rates.


Assuntos
Regiões 3' não Traduzidas/genética , Bactérias/genética , Códon de Terminação/genética , Códon sem Sentido/genética , Eucariotos/genética , Evolução Molecular , Expressão Gênica/genética , Genoma , Degradação do RNAm Mediada por Códon sem Sentido/genética , Fases de Leitura Aberta/genética
6.
Amino Acids ; 51(9): 1273-1288, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31440819

RESUMO

The multifunctional protein Transglutaminase type 2, is associated with cancer epithelial mesenchymal transition, invasiveness, stemness and drugs resistance. Several variant isoforms and non-coding RNAs are present in cancer and this report explored the expression of these transcripts of the TGM2 gene in cancer cell lines after induction with all-trans retinoic acid. The expression of truncated variants along with two long non-coding RNAs, was demonstrated. One of these is coded from the first intron and the Last Exon Variant is constituted by a sequence corresponding to the last three exons and the 3'UTR. Analysis of ChIP-seq data, from ENCODE project, highlighted factors interacting with intronic sequences, which could interfere with the progression of RNApol II at checkpoints, during the elongation process. Some relevant transcription factors, bound in an ATRA-dependent way, were found by RNA immunoprecipitation, notably GATA3 mainly enriched to Last Exon Variant non-coding RNA. The involvement of NMD in the regulation of the ratio among these transcripts was observed, as the prevalent recovering of Last Exon Variant to phUPF1-complexes, with decrease of the binding towards other selective targets. This study contributes to identify molecular mechanisms regulating the ratio among the variants and improves the knowledge about regulatory roles of the non-coding RNAs of the TGM2 gene.


Assuntos
Proteínas de Ligação ao GTP/biossíntese , RNA Longo não Codificante/metabolismo , Transglutaminases/biossíntese , Tretinoína/farmacologia , Fator de Transcrição GATA3/metabolismo , Proteínas de Ligação ao GTP/genética , Células HL-60 , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Degradação do RNAm Mediada por Códon sem Sentido , RNA Longo não Codificante/genética , Transcrição Genética , Transglutaminases/genética
7.
Plant Cell Physiol ; 60(9): 1986-1999, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31368494

RESUMO

Nonsense-mediated decay (NMD) is an RNA surveillance mechanism that detects aberrant transcript features and triggers degradation of erroneous as well as physiological RNAs. Originally considered to be constitutive, NMD is now recognized to be tightly controlled in response to inherent signals and diverse stresses. To gain a better understanding of NMD regulation and its functional implications, we systematically examined feedback control of the central NMD components in two dicot and one monocot species. On the basis of the analysis of transcript features, turnover rates and steady-state levels, up-frameshift (UPF) 1, UPF3 and suppressor of morphological defects on genitalia (SMG) 7, but not UPF2, are under feedback control in both dicots. In the monocot investigated in this study, only SMG7 was slightly induced upon NMD inhibition. The detection of the endogenous NMD factor proteins in Arabidopsis thaliana substantiated a negative correlation between NMD activity and SMG7 amounts. Furthermore, evidence was provided that SMG7 is required for the dephosphorylation of UPF1. Our comprehensive and comparative study of NMD feedback control in plants reveals complex and species-specific attenuation of this RNA surveillance pathway, with critical implications for the numerous functions of NMD in physiology and stress responses.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Retroalimentação Fisiológica , Degradação do RNAm Mediada por Códon sem Sentido , Estabilidade de RNA , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica de Plantas , RNA Helicases/genética , RNA Helicases/metabolismo , RNA de Plantas/genética , Especificidade da Espécie
8.
Nucleic Acids Res ; 47(17): 9216-9230, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31428786

RESUMO

XRN4, the plant cytoplasmic homolog of yeast and metazoan XRN1, catalyzes exoribonucleolytic degradation of uncapped mRNAs from the 5' end. Most studies of cytoplasmic XRN substrates have focused on polyadenylated transcripts, although many substrates are likely first deadenylated. Here, we report the global investigation of XRN4 substrates in both polyadenylated and nonpolyadenylated RNA to better understand the impact of the enzyme in Arabidopsis. RNA degradome analysis demonstrated that xrn4 mutants overaccumulate many more decapped deadenylated intermediates than those that are polyadenylated. Among these XRN4 substrates that have 5' ends precisely at cap sites, those associated with photosynthesis, nitrogen responses and auxin responses were enriched. Moreover, xrn4 was found to be defective in the dark stress response and lateral root growth during N resupply, demonstrating that XRN4 is required during both processes. XRN4 also contributes to nonsense-mediated decay (NMD) and xrn4 accumulates 3' fragments of select NMD targets, despite the lack of the metazoan endoribonuclease SMG6 in plants. Beyond demonstrating that XRN4 is a major player in multiple decay pathways, this study identified intriguing molecular impacts of the enzyme, including those that led to new insights about mRNA decay and discovery of functional contributions at the whole-plant level.


Assuntos
Arabidopsis/genética , Exorribonucleases/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Proteínas de Plantas/genética , Estabilidade de RNA/genética , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Nitrogênio/metabolismo , RNA Mensageiro/genética
9.
Plant Cell Physiol ; 60(9): 2000-2014, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31386149

RESUMO

Plants generally possess a strong ability to regenerate organs; for example, in tissue culture, shoots can regenerate from callus, a clump of actively proliferating, undifferentiated cells. Processing of pre-mRNA and ribosomal RNAs is important for callus formation and shoot regeneration. However, our knowledge of the roles of RNA quality control via the nonsense-mediated mRNA decay (NMD) pathway in shoot regeneration is limited. Here, we examined the shoot regeneration phenotypes of the low-beta-amylase1 (lba1)/upstream frame shift1-1 (upf1-1) and upf3-1 mutants, in which the core NMD components UPF1 and UPF3 are defective. These mutants formed callus from hypocotyl explants normally, but this callus behaved abnormally during shoot regeneration: the mutant callus generated numerous adventitious root structures instead of adventitious shoots in an auxin-dependent manner. Quantitative RT-PCR and microarray analyses showed that the upf mutations had widespread effects during culture on shoot-induction medium. In particular, the expression patterns of early auxin response genes, including those encoding AUXIN/INDOLE ACETIC ACID (AUX/IAA) family members, were significantly affected in the upf mutants. Also, the upregulation of shoot apical meristem-related transcription factor genes, such as CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, was inhibited in the mutants. Taken together, these results indicate that NMD-mediated transcriptomic regulation modulates the auxin response in plants and thus plays crucial roles in the early stages of shoot regeneration.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Degradação do RNAm Mediada por Códon sem Sentido , Reguladores de Crescimento de Planta/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Hipocótilo/genética , Hipocótilo/fisiologia , Ácidos Indolacéticos/metabolismo , Meristema/genética , Meristema/fisiologia , Mutação , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/fisiologia , Transdução de Sinais
10.
Int J Mol Sci ; 20(17)2019 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-31450582

RESUMO

Mutations in the CDKL5 gene lead to an incurable rare neurological condition characterized by the onset of seizures in the first weeks of life and severe intellectual disability. Replacement gene or protein therapies could represent intriguing options, however, their application may be inhibited by the recent demonstration that CDKL5 is dosage sensitive. Conversely, correction approaches acting on pre-mRNA splicing would preserve CDKL5 physiological regulation. Since ~15% of CDKL5 pathogenic mutations are candidates to affect splicing, we evaluated the capability of variants of the spliceosomal U1 small nuclear RNA (U1snRNA) to correct mutations affecting +1 and +5 nucleotides at the 5' donor splice site and predicted to cause exon skipping. Our results show that CDKL5 minigene variants expressed in mammalian cells are a valid approach to assess CDKL5 splicing pattern. The expression of engineered U1snRNA effectively rescued mutations at +5 but not at the +1 nucleotides. Importantly, we proved that U1snRNA-mediated splicing correction fully restores CDKL5 protein synthesis, subcellular distribution and kinase activity. Eventually, by correcting aberrant splicing of an exogenously expressed splicing-competent CDKL5 transgene, we provided insights on the morphological rescue of CDKL5 null neurons, reporting the first proof-of-concept of the therapeutic value of U1snRNA-mediated CDKL5 splicing correction.


Assuntos
Mutação , Proteínas Serina-Treonina Quinases/genética , Processamento de RNA , RNA Nuclear Pequeno/genética , Reparo Gênico Alvo-Dirigido , Alelos , Processamento Alternativo , Linhagem Celular , Síndromes Epilépticas/genética , Síndromes Epilépticas/terapia , Éxons , Expressão Gênica , Loci Gênicos , Terapia Genética , Genótipo , Humanos , Neurônios/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas Serina-Treonina Quinases/metabolismo , Espasmos Infantis/genética , Espasmos Infantis/terapia
11.
Nucleic Acids Res ; 47(16): 8838-8859, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31329944

RESUMO

Regnase-1-mediated mRNA decay (RMD), in which inflammatory mRNAs harboring specific stem-loop structures are degraded, is a critical part of proper immune homeostasis. Prior to initial translation, Regnase-1 associates with target stem-loops but does not carry out endoribonucleolytic cleavage. Single molecule imaging revealed that UPF1 is required to first unwind the stem-loops, thus licensing Regnase-1 to proceed with RNA degradation. Following translation, Regnase-1 physically associates with UPF1 using two distinct points of interaction: The Regnase-1 RNase domain binds to SMG1-phosphorylated residue T28 in UPF1; in addition, an intrinsically disordered segment in Regnase-1 binds to the UPF1 RecA domain, enhancing the helicase activity of UPF1. The SMG1-UPF1-Regnase-1 axis targets pioneer rounds of translation and is critical for rapid resolution of inflammation through restriction of the number of proteins translated by a given mRNA. Furthermore, small-molecule inhibition of SMG1 prevents RNA unwinding in dendritic cells, allowing post-transcriptional control of innate immune responses.


Assuntos
Macrófagos Peritoneais/imunologia , Degradação do RNAm Mediada por Códon sem Sentido/imunologia , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Ribonucleases/genética , Transativadores/genética , Animais , Fibroblastos/citologia , Fibroblastos/imunologia , Células HEK293 , Células HeLa , Homeostase/genética , Homeostase/imunologia , Humanos , Imunidade Inata , Inflamação , Sequências Repetidas Invertidas , Macrófagos/citologia , Macrófagos/imunologia , Macrófagos Peritoneais/citologia , Camundongos , Camundongos Knockout , Mutação , Cultura Primária de Células , Ligação Proteica , Biossíntese de Proteínas , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/imunologia , RNA Mensageiro/metabolismo , Ribonucleases/deficiência , Ribonucleases/imunologia , Imagem Individual de Molécula , Transativadores/imunologia
12.
Cells ; 8(7)2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31330982

RESUMO

Moso bamboo is an important forest species with a variety of ecological, economic, and cultural values. However, the gene annotation information of moso bamboo is only based on the transcriptome sequencing, lacking the evidence of proteome. The lignification and fiber in moso bamboo leads to a difficulty in the extraction of protein using conventional methods, which seriously hinders research on the proteomics of moso bamboo. The purpose of this study is to establish efficient methods for extracting the total proteins from moso bamboo for following mass spectrometry-based quantitative proteome identification. Here, we have successfully established a set of efficient methods for extracting total proteins of moso bamboo followed by mass spectrometry-based label-free quantitative proteome identification, which further improved the protein annotation of moso bamboo genes. In this study, 10,376 predicted coding genes were confirmed by quantitative proteomics, accounting for 35.8% of all annotated protein-coding genes. Proteome analysis also revealed the protein-coding potential of 1015 predicted long noncoding RNA (lncRNA), accounting for 51.03% of annotated lncRNAs. Thus, mass spectrometry-based proteomics provides a reliable method for gene annotation. Especially, quantitative proteomics revealed the translation patterns of proteins in moso bamboo. In addition, the 3284 transcript isoforms from 2663 genes identified by Pacific BioSciences (PacBio) single-molecule real-time long-read isoform sequencing (Iso-Seq) was confirmed on the protein level by mass spectrometry. Furthermore, domain analysis of mass spectrometry-identified proteins encoded in the same genomic locus revealed variations in domain composition pointing towards a functional diversification of protein isoform. Finally, we found that part transcripts targeted by nonsense-mediated mRNA decay (NMD) could also be translated into proteins. In summary, proteomic analysis in this study improves the proteomics-assisted genome annotation of moso bamboo and is valuable to the large-scale research of functional genomics in moso bamboo. In summary, this study provided a theoretical basis and technical support for directional gene function analysis at the proteomics level in moso bamboo.


Assuntos
Genoma de Planta , Proteínas de Plantas , Poaceae/genética , Estabilidade de RNA , Processamento Alternativo , Anotação de Sequência Molecular , Degradação do RNAm Mediada por Códon sem Sentido , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Isoformas de Proteínas/genética , Proteogenômica , RNA Longo não Codificante
13.
Adv Exp Med Biol ; 1157: 41-83, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31342437

RESUMO

Nonsense-mediated mRNA decay (NMD) is a well characterized eukaryotic mRNA degradation pathway, responsible for the identification and degradation of transcripts harboring translation termination codons in premature contexts. Transcriptome-wide studies revealed that NMD is not only an mRNA surveillance pathway as initially thought, but is also a post-transcriptional regulatory mechanism of gene expression, as it fine-tunes the transcript levels of many wild-type genes. Hence, NMD contributes to the regulation of many essential biological processes, including pathophysiological mechanisms. In this chapter we discuss the importance of NMD and of its regulation to organism development and its link to the cellular stress responses, like the unfolded protein response (UPR) and the integrated stress response (ISR). Additionally, we describe how tumor cells have explored both NMD functions to promote tumorigenesis. Using published data and databases, we have also performed a network-based approach that further supports the link between NMD and these (patho) physiological processes.


Assuntos
Neoplasias , Degradação do RNAm Mediada por Códon sem Sentido , Humanos , Neoplasias/fisiopatologia , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Transcriptoma
14.
Nature ; 572(7769): 335-340, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31316208

RESUMO

Lamin A/C (LMNA) is one of the most frequently mutated genes associated with dilated cardiomyopathy (DCM). DCM related to mutations in LMNA is a common inherited cardiomyopathy that is associated with systolic dysfunction and cardiac arrhythmias. Here we modelled the LMNA-related DCM in vitro using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Electrophysiological studies showed that the mutant iPSC-CMs displayed aberrant calcium homeostasis that led to arrhythmias at the single-cell level. Mechanistically, we show that the platelet-derived growth factor (PDGF) signalling pathway is activated in mutant iPSC-CMs compared to isogenic control iPSC-CMs. Conversely, pharmacological and molecular inhibition of the PDGF signalling pathway ameliorated the arrhythmic phenotypes of mutant iPSC-CMs in vitro. Taken together, our findings suggest that the activation of the PDGF pathway contributes to the pathogenesis of LMNA-related DCM and point to PDGF receptor-ß (PDGFRB) as a potential therapeutic target.


Assuntos
Cardiomiopatia Dilatada/genética , Lamina Tipo A/genética , Mutação , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/patologia , Cálcio/metabolismo , Células Cultivadas , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Haploinsuficiência/genética , Homeostase , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/patologia , Modelos Biológicos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Degradação do RNAm Mediada por Códon sem Sentido , RNA Mensageiro/análise , RNA Mensageiro/genética , Análise de Célula Única
15.
Mol Cell ; 75(2): 324-339.e11, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31155380

RESUMO

Nonsense-mediated decay (NMD) is a surveillance system that degrades mRNAs containing a premature termination codon (PTC) and plays important roles in protein homeostasis and disease. The efficiency of NMD is variable, impacting the clinical outcome of genetic mutations. However, limited resolution of bulk analyses has hampered the study of NMD efficiency. Here, we develop an assay to visualize NMD of individual mRNA molecules in real time. We find that NMD occurs with equal probability during each round of translation of an mRNA molecule. However, this probability is variable and depends on the exon sequence downstream of the PTC, the PTC-to-intron distance, and the number of introns both upstream and downstream of the PTC. Additionally, a subpopulation of mRNAs can escape NMD, further contributing to variation in NMD efficiency. Our study uncovers real-time dynamics of NMD, reveals key mechanisms that influence NMD efficiency, and provides a powerful method to study NMD.


Assuntos
Códon sem Sentido/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , RNA Mensageiro/genética , Códon sem Sentido/química , Éxons/genética , Humanos , Íntrons/genética , Mutação/genética , Estabilidade de RNA/genética , RNA Mensageiro/química , Imagem Individual de Molécula
16.
Chimia (Aarau) ; 73(6): 374-379, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31118119

RESUMO

Eukaryotic cells have evolved a number of survival tactics and quality control pathways that are able to counter intrinsic error-prone mechanisms and allow for maintenance of cellular homeostasis in the face of external stresses. This review will discuss the role of two cellular eukaryotic processes that are vital for maintenance of cellular homeostasis - 1) the nonsense-mediated mRNA decay (NMD) pathway and 2) the transient formation of stress granules (SG) - and explore the current literature on their roles in antiviral defence. Within the NCCR RNA & Disease, the laboratories of Proffs. O. Mühlemann and Volker Thiel teamed up to unravel the roles of NMD and SGs, and their interconnections in defending cells from alphavirus and coronavirus infections.


Assuntos
Degradação do RNAm Mediada por Códon sem Sentido , Antivirais , RNA
17.
Plant Cell Physiol ; 60(9): 1953-1960, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31111919

RESUMO

Post-transcriptional RNA quality control is a vital issue for all eukaryotes to secure accurate gene expression, both on a qualitative and quantitative level. Among the different mechanisms, nonsense-mediated mRNA decay (NMD) is an essential surveillance system that triggers degradation of both aberrant and physiological transcripts. By targeting a substantial fraction of all transcripts for degradation, including many alternative splicing variants, NMD has a major impact on shaping transcriptomes. Recent progress on the transcriptome-wide profiling and physiological analyses of NMD-deficient plant mutants revealed crucial roles for NMD in gene regulation and environmental responses. In this review, we will briefly summarize our current knowledge of the recognition and degradation of NMD targets, followed by an account of NMD's regulation and physiological functions. We will specifically discuss plant-specific aspects of RNA quality control and its functional contribution to the fitness and environmental responses of plants.


Assuntos
Processamento Alternativo/genética , Regulação da Expressão Gênica de Plantas/genética , Degradação do RNAm Mediada por Códon sem Sentido/genética , Plantas/genética , Transcriptoma , Perfilação da Expressão Gênica , Fenômenos Fisiológicos Vegetais , Especificidade da Espécie
18.
Nucleic Acids Res ; 47(12): 6330-6338, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31045216

RESUMO

In-frame stop codons mark the termination of translation. However, post-termination ribosomes can reinitiate translation at downstream AUG codons. In mammals, reinitiation is most efficient when the termination codon is positioned close to the 5'-proximal initiation site and around 78 bases upstream of the reinitiation site. The phenomenon was studied mainly in the context of open reading frames (ORFs) found within the 5'-untranslated region, or polycicstronic viral mRNA. We hypothesized that reinitiation of translation following nonsense mutations within the main ORF of p53 can promote the expression of N-truncated p53 isoforms such as Δ40, Δ133 and Δ160p53. Here, we report that expression of all known N-truncated p53 isoforms by reinitiation is mechanistically feasible, including expression of the previously unidentified variant Δ66p53. Moreover, we found that significant reinitiation of translation can be promoted by nonsense mutations located even 126 codons downstream of the 5'-proximal initiation site, and observed when the reinitiation site is positioned between 6 and 243 bases downstream of the nonsense mutation. We also demonstrate that reinitiation can stabilise p53 mRNA transcripts with a premature termination codon, by allowing such transcripts to evade the nonsense mediated decay pathway. Our data suggest that the expression of N-truncated proteins from alleles carrying a premature termination codon is more prevalent than previously thought.


Assuntos
Códon sem Sentido , Iniciação Traducional da Cadeia Peptídica , Proteína Supressora de Tumor p53/genética , Linhagem Celular , Células HEK293 , Humanos , Degradação do RNAm Mediada por Códon sem Sentido , Regiões Promotoras Genéticas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Deleção de Sequência , Proteína Supressora de Tumor p53/biossíntese
19.
Mol Cells ; 42(4): 301-312, 2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31091556

RESUMO

Post-transcriptional regulation underlies the circadian control of gene expression and animal behaviors. However, the role of mRNA surveillance via the nonsense-mediated mRNA decay (NMD) pathway in circadian rhythms remains elusive. Here, we report that Drosophila NMD pathway acts in a subset of circadian pacemaker neurons to maintain robust 24 h rhythms of free-running locomotor activity. RNA interference-mediated depletion of key NMD factors in timeless-expressing clock cells decreased the amplitude of circadian locomotor behaviors. Transgenic manipulation of the NMD pathway in clock neurons expressing a neuropeptide PIGMENT-DISPERSING FACTOR (PDF) was sufficient to dampen or lengthen free-running locomotor rhythms. Confocal imaging of a transgenic NMD reporter revealed that arrhythmic Clock mutants exhibited stronger NMD activity in PDF-expressing neurons than wild-type. We further found that hypomorphic mutations in Suppressor with morphogenetic effect on genitalia 5 (Smg5 ) or Smg6 impaired circadian behaviors. These NMD mutants normally developed PDF-expressing clock neurons and displayed daily oscillations in the transcript levels of core clock genes. By contrast, the loss of Smg5 or Smg6 function affected the relative transcript levels of cAMP response element-binding protein B (CrebB ) in an isoform-specific manner. Moreover, the overexpression of a transcriptional repressor form of CrebB rescued free-running locomotor rhythms in Smg5-depleted flies. These data demonstrate that CrebB is a rate-limiting substrate of the genetic NMD pathway important for the behavioral output of circadian clocks in Drosophila.


Assuntos
Relógios Circadianos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Mutação , Degradação do RNAm Mediada por Códon sem Sentido , Transativadores/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas CLOCK/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Neurônios/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , Transdução de Sinais
20.
Yi Chuan ; 41(5): 359-364, 2019 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-31106771

RESUMO

The genetic compensation response (GCR) was firstly described in zebrafish to explain the phenotypic discrepancies between gene-knockout and gene-knockdown, whereby a deleterious mutation, but not gene-knockdown, can lead to the transcriptional upregulation of related genes, which can assume the function of the mutated gene. This phenomenon was also found in other model systems including mice and Arabidopsis. However, the underlying molecular mechanism of the GCR remains elusive until two papers were published in Nature on April 3, 2019: one from our lab and the other from Stainier's lab. Using different genetic mutants of various genes in zebrafish or culture cells of mice, both of us reveal that the upregulation of compensatory genes is only triggered by mutations that generate a premature termination codon (PTC); the compensatory genes share nucleotide sequence homology to the mutated genes; nonsense mRNA mediated decay pathway (NMD) is essential for the induction of GCR, and the increased transcription of the compensatory genes is accompanied by an enhancement of H3K4 trimethylation (H3K4me3) at their transcription start site (TSS) regions. In this review, we summarize the mechanisms of the GCR proposed in the two studies.


Assuntos
Códon sem Sentido , Degradação do RNAm Mediada por Códon sem Sentido , Ativação Transcricional , Animais , Histonas , Metilação , Camundongos , Mutação , RNA Mensageiro/genética , Sítio de Iniciação de Transcrição , Peixe-Zebra
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